Central nervous system is protected by the blood-brain barrier, which represents a physical, metabolic and transport barrier and is considered to be a part of a highly dynamic system termed neurovascular unit. Several pathogens, among them viruses, are able to invade the brain. Traversal of viruses across the blood-brain barrier is an essential step for the invasion of the central nervous system and can occur by different mechanisms - by paracellular, transcellular and/or by "Trojan horse" pathway. Penetration of viruses to brain can lead to the blood-brain barrier dysfunction, including increased permeability, pleocytosis and encephalopathy. Viruses causing the central nervous system infections include human immunodeficiency virus type 1, rhabdovirus, different flaviviruses, mouse adenovirus type 1, herpes simplex virus, influenza virus, parainfluenza virus, reovirus, lymphocytic choriomeningitis virus, arbovirus, cytomegalovirus, mumps virus, parvovirus B19, measles virus, human T-cell leukemia virus, enterovirus, morbillivirus, bunyaviruses, togaviruses and others. In this review we summarized what is known about the routes of how some viruses enter the brain and how neurons and glial cells react to infection.
The mechanisms by which Streptococcus pneumoniae penetrates the blood-brain barrier (BBB), reach the CNS and causes meningitis are not fully understood. Adhesion of bacterial cells on the brain microvascular endothelial cells (BMECs), mediated through protein-protein interactions, is one of the crucial steps in translocation of bacteria across BBB. In this work, we proposed a systematic workflow for identification of cell wall associated ligands of pneumococcus that might adhere to the human BMECs. The proteome of S. pneumoniae was biotinylated and incubated with BMECs. Interacting proteins were recovered by affinity purification and identified by data independent acquisition (DIA). A total of 44 proteins were identified from which 22 were found to be surface-exposed. Based on the subcellular location, ontology, protein interactive analysis and literature review, five ligands (adhesion lipoprotein, endo-β-N-acetylglucosaminidase, PhtA and two hypothetical proteins, Spr0777 and Spr1730) were selected to validate experimentally (ELISA and immunocytochemistry) the ligand-BMECs interaction. In this study, we proposed a high-throughput approach to generate a dataset of plausible bacterial ligands followed by systematic bioinformatics pipeline to categorize the protein candidates for experimental validation. The approach proposed here could contribute in the fast and reliable screening of ligands that interact with host cells.
Ligand-receptor interactions play a crucial role in the plethora of biological processes. Several methods have been established to reveal ligand-receptor interface, however, the majority of methods are time-consuming, laborious and expensive. Here we present a straightforward and simple pipeline to identify putative receptor-binding sites on the pathogen ligands. Two model ligands (bait proteins), domain III of protein E of West Nile virus and NadA of Neisseria meningitidis, were incubated with the proteins of human brain microvascular endothelial cells immobilized on nitrocellulose or PVDF membrane, the complex was trypsinized on-membrane, bound peptides of the bait proteins were recovered and detected on MALDI-TOF. Two peptides of DIII (~916 Da and ~2003 Da) and four peptides of NadA (~1453 Da, ~1810 Da, ~2051 Da and ~2433 Da) were identified as plausible receptor-binders. Further, binding of the identified peptides to the proteins of endothelial cells was corroborated using biotinylated synthetic analogues in ELISA and immunocytochemistry. Experimental pipeline presented here can be upscaled easily to map receptor-binding sites on several ligands simultaneously. The approach is rapid, cost-effective and less laborious. The proposed experimental pipeline could be a simpler alternative or complementary method to the existing techniques used to reveal amino-acids involved in the ligand-receptor interface.
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